Cytoplasmic Ca2+ increases triggered by activation of inositol trisphosphate receptors regulate a huge variety of physiological events. Dysfunction of the same IP3-stimulated Ca2+ release pathway underlies the pathogenesis of many disorders. Therefore, it is important to understand how IP3 receptors generate physiologically relevant Ca2+ signals, and how pathological events dysregulate IP3 receptor behavior. Our understanding of the functional organization of IP3-evoked Ca2+ signals in intact cells has been transformed by high resolution imaging methods. Confocal imaging has shown that IP3-evoked Ca2+ signals are generated by progressive recruitment of microscopic Ca2+ signals, known as 'Ca2+ puffs', which are resolved as transient changes in fluorescence of high affinity Ca2+ indicator dyes. These functional signals are interpreted as reflecting the activity of small clusters of IP3 receptors spaced throughout the endoplasmic reticulum, but the underlying distribution, and structural architecture of IP3 receptors is unresolved. As spatial patterning of whole cell Ca2+ signals depends on both the function and the cellular distribution of Ca2+ release channels, it is imperative to understand the mechanisms that control the 'functional architecture' of IP3 receptors. The aim of this proposal is to define how IP3 receptor distribution impacts the patterning of cellular Ca2+ signals to test the central hypothesis that IP3 receptor architecture is modulated by physiological and pathological cues. To achieve this goal, we have optimized fluorescent protein-based tools that resolve IP3 receptor distribution in live cells. We will use these novel tools, as well as biochemical and molecular approaches to (1) resolve IP3 receptor architecture underlying different spatiotemporal patterns of 1P3-evoked Ca2+ signaling; (2) define the mechanisms of physiological change in the functional architecture of lP3 receptor signaling during early development and (3) delineate the mechanisms of pathological change in the functional architecture of lP3 receptor signaling evoked by the hepatitis C viral protein NS5A. Results will aid our understanding of the role of the ubiquitous IP3 signaling pathway in both health and disease.